Platelet Review July 2012 Thomas S. Kickler M.D. Johns Hopkins University School of Medicine
Hemostasis • Hemostasis is the process that leads to the stopping of bleeding • Hemostasis involves blood vessels, platelets, plasma clotting proteins • Primary Hemostasis is the initial response to injury to a blood vessel involving platelets • Secondary Hemostasis occurs to fortify primary hemostasis thru the activation of clotting proteins to form a insoluble deposition of fibrin in and around platelets
Bleeding – Cut a Blood Vessel – What Happens ?
What is Going on in the Blood Vessel – a lot! SECONDARY HEMOSTASIS = CLOTTING CASCADE Platelet PRIMARY HEMOSTASIS = PLATELET PLUG
Overview of Hemostasis Clotting Cascade Leads to Secondary Hemostasis Platelets Lead to Primary Hemostasis Hemostasis- 2 components- Platelets, Clotting Proteins Both Occurring Simultaneously
Hemostasis • Intricate system maintaining blood in fluid state – Reacts to vascular injury to stop blood loss and seal vessel wall • Involves platelets, clotting factors, endothelium, and inhibitory/control mechanisms – Highly developed system of checks and balances Normal Hemostasis Absence of overt bleeding/thrombosis Bleeding Thrombosis
Platelets are typically 1-2 micron The normal PLT count is 150-350,000/ul One large one above shows how granular they appear.
A scanning electron micrograph of normal platelets Really are fragments of megakaryocyte cytoplasm
Platelet- Number, Lifespan and Kinetics • Normal platelet concentration is 150,000- 350,000/ ul • Platelets are produced in the bone marrow by megakaryocytes and released into the circulation • They circulate in the blood for about 10 days after release from the marrow • About 1/3 of all the body’s platelet mass is stored in the spleen
Megakaryocytes produce platelets in the marrow, stimulated by thrombopoietin
Normal Megakaryocyte Platelets are released from megakaryocytes , this shows this process in vitro culture
Hematopoiesis T cells lymphocytes B cells monocyte neutrophil Stem cell RBC platelets
Stages of platelet development Terminal commitment differentiation Stem cell BFU-Mk Immature Mk CFU-Mk Mature Mk All stages are driven by thrombopoietin Platelet shedding
Thrombopoietin (TPO) • Growth factor produced in liver • Increases production of megakaryocytes • Essential for stem cells
control +TPO TPO Effect on Normal Mice 7 6 5 4 Days 3 2 1 0 6 5 4 3 2 1 0 6 /mm 3 ) Platelets ( x 10
+TPO Mouse bone marrow control
TPO regulation • Constitutive (constant) production • Level depends on binding sites on platelets and megakaryocytes
Thrombopoietin Regulation (Sponge theory) TPO PLT PLT MPL As Platelet Count Increases, serve as a sponge , having less available to stimulate Megakaryocytes
Primary Hemostasis Aggregation Adhesion Secretion
Adhesion occurs within 1-3 seconds after injury
As adhesion occurs, platelets release ADP and Thromboxane (TxA2) , these help recruit other platelets into the platelet plug and as secondary hemostasis gets started thombin is generated, causing more platelet stimulation and conversion of fibrinogen to fibrin
3-7 minutes for entire process to occur-”The Bleeding Time”
Activated platelets Note pseudopodia and how platelets aggregating to each other
A scanning EM of a clot with platelets, RBCs trapped in mesh of developing fibrin
Remember Platelets act in Concert Thrombin with Fibrin Formation to Form a Firm transforms Clot fibrinogen to a Fibrinogen mesh of fibrin strands Fibrin – polymerized remains of fibrinogen EM of Think of fibrin as fibrinogen strands of protein that that has been holds the platelets treated with together Thombin
Summary of Platelet Processess
Testing for Abnormal Platelet Function Bleeding Time Normal 3-7 minutes Prolonged in platelet function abnormalities
Bleeding Time Aggregometry optical density time An abnormal response to ADP normal < 7-8min A bleeding time that did not
Bleeding – Cut a Blood Vessel – What Happens ?
The Endothelium Prevents Excess Platelet Function In Vivo The endothelium is “antagonistic” to platelets under normal conditions
Vascular Endothelium Function Anticoagulant- Inhibits coagulation Tissue factor pathway extrinsic pathway inhibitor Anticoagulant- Inhibits coagulation by Thrombomodulin activating protein C system Anticoagulant- Inhibits coagulation by Tissue plasminogen activator activating fibrinolysis Anticoagulant- Inhibits coagulation by Heparan sulfate proteoglycans activating antithrombin Tissue factor Procoagulant- Inflammatory cytokines (IL-1, TNF) induce expression
Vascular Endothelium Function Vasodilation, inhibition of platelet Prostacyclin aggregation From platelets, muscular arteries constrict Thromboxane A 2 Cytokines induce synthesis to promote leukocyte adhesion ELAMs, ICAMs Promote platelet-collagen adhesion to exposed sub-endothelium von Willebrand factor
Thombocytopenia • > 100,000/ul no excessive bleeding, even with major surgery • 50-100,000 may bleed longer than normal with severe trauma • 20-50,000 bleed with minor trauma • < 20,000 may have spontaneous hemorrhage
Petechiae- subcutaneous bleeding develops when the platelet counts falls below 20- 50, 000/ul
Adherens Junction at the Postcapillary Venular Bed Nachman R and Rafii S. N Engl J Med 2008;359:1261-1270
Bleeding in Patients with Thrombocytopenia through Disassembly of the Adherens Junction Nachman R and Rafii S. N Engl J Med 2008;359:1261-1270
Causes of Thrombocytopenia • Decreased production: marrow hypoplasia, leukemia, toxins, chemotherapy • Increased Destruction: antibodies to platelets, activation of coagulation cascade resulting in PLT consumption • Platelet Sequestration: 1/3 of platelets are normally stored in the spleen, if enlarges more platelets are stored and patient becomes thrombocytopenic
Decreased Production No straightforward method to Decreased production: marrow hypoplasia, leukemia, toxins, assess platelet production , unlike chemotherapy RBCs & Retic Count
Severe thrombocytopenia in Autoimmune thrombocytopenia Blood smear shows no platelets Isotope labeled platelets are destroyed in the spleen, in presence of antibody
Pathophysiology of Autoimmune Thrombocytopenia An example of a Autoantibodies are formed against the platelet common glycoprotein receptor IIb-IIIa, and are destroyed in the consumptive Reticuloendothelial system thrombocytopenia
Panel A , patient without increased Megakaryocytes, versus patients with increased megakaryocytes
Overview of Immature Platelet Fraction Percentage (IPF%) Measurement & Some Examples normal ITP H-IPF(%) (Logarithm ) Ref: 1% FSC (Cell size) IPF(%) Ref: 3% IPF# (x10 9 /L) Fluorescence intensity (Linear ) PLT-X (ch) Nadir after Recovery from Chemo Chemo
Thrombocytosis Seen in myeloproliferative disorders, chronic infection, iron deficiencies, malignant tumors
Platelets Role in Thrombosis • Coronary or cerebrovascular thrombosis is multifactorial • Genes – lipids • Society – diet, exercise, smoking
Triggers of Thrombosis Artery v Vein ARTERY – VEIN – CLOTTING PLATELETS PROTEINS ROLE ROLE CERTAIN CERTAIN
Summary • Describe the major physiologic functions of platelets • Describe the major platelet agonists • Describe the ligands responsible for adhesion and aggregation • Describe the pathophysiology of thrombocytopenia
Hemostasis- Summary
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